MAPK信號傳導通路的調(diào)控對皮膚光老化的保護作用研究
發(fā)布時間：2018-09-17 07:29
【摘要】：目的研究UVA照射體外培養(yǎng)的皮膚成纖維細胞后,細胞形態(tài)變化及相關細胞因子變化,檢測MAPK信號傳導的始動受體EGFR的表達情況,及MAPK信號途徑中的下游基因c-jun,c-fos的表達變化。通過篩選出高效特異的c-junsiRNA后,對UVA照射后的皮膚成纖維細胞進行轉(zhuǎn)染,探討下調(diào)MAPK下游的基因c-jun對皮膚光老化中的MMPs,膠原的影響。 方法：1.取年輕成人包皮成纖維細胞培養(yǎng)傳代,用第5代細胞做實驗對象,進行細胞分組,采用不同計量的UVA(5 J/cm2、10J/cm2、20J/cm2)照射,設立對照組(0J/cm2),建立成纖維細胞光老化模型,培養(yǎng)24h后,光鏡及電鏡下觀察細胞形態(tài),RT-PCR及酶聯(lián)免疫法(ELISA)測定照射前后體外培養(yǎng)的皮膚成纖維細胞的Ⅰ、Ⅲ型前膠原mRNA表達及蛋白表達。MTT法測定細胞活性。2.采用UVA(5 J/cm210J/cm2,15J/cm2)照射,設立對照組,照射后24小時用realtimePCR檢測MAPK信號傳導途徑下游基因c-jun,c-fos,及終末基因MMP-1,MMP-3的mRNA表達情況。酶聯(lián)免疫法(ELISA)測定細胞上清液中的MMP-1,MMP-3表達,weasternblog檢測c-jun, c-fos的表達。3.針對前期實驗中c-jun隨照射劑量依賴性增高,設計5條c-junsiRNA, realtimePCR,及westembolg從基因?qū)用婧偷鞍讓用鎸ζ浜Y選,篩選出沉默效率最高的c-junsiRAN,并優(yōu)化轉(zhuǎn)染條件。4。以15J/cm2輻射劑量照射體外培養(yǎng)的成纖維細胞,2小時后轉(zhuǎn)染c-junsiRNA,轉(zhuǎn)染后24小時,realtimePCR檢測MMP-1、MMP-3 mRNA,Ⅰ、Ⅲ型前膠原mRNA,48小時后酶聯(lián)免疫法(ELISA)檢測細胞上清液中MMP-1、MMP-3,Ⅰ、Ⅲ型前膠原含量。5,以(5 J/cm2、10J/cm2、20J/cm2)照射成纖維細胞,設立對照,檢測MAPK信號傳導通路的始動受體EGFR的表達情況,檢測TGF-β1、IGF-1、KGF、VEGF幾個細胞因子在照射前后的變化. 結果：1.隨著UVA照射劑量增加,成纖維細胞MTT的OD值下降,與對照組比較,UVA 10 J/cm2、UVA 20 J/cm2照射組OD值明顯下降,有顯著性差異(p0.01)。Ⅰ型(?)mRNA RT-PCR產(chǎn)物積光吸光度隨著UVA照射劑量增加而降低,UVA 10 J/cm2、UVA20J/cm2照射組降低明顯(p0.05,P0.01),UVA 20 J/cm2組抑制膠原合成作用更加明顯,與對照組及其它兩組比較,有非常顯著性差異(P0.01)。Ⅲ型膠原含量和mRNA表達,與對照組比較,UVA 20 J/cm2照射組明顯降低,有非常顯著性差異(P0.01)。細胞上清液中Ⅰ型膠原含量隨著UVA照射劑量增加而降低,呈劑量依賴關系。UVA 10J/cm2、UVA 20J/cm2照射組,Ⅰ型膠原含量明顯降低,與對照組比較(P0.05, P0.01),說明成纖維細胞分別給予UVA 10J/cm2、UVA 20J/cm2照射劑量,均可以抑制膠原合成,且劑量UVA 20 J/cm2組抑制膠原合成作用更加明顯,與對照組及其它兩組比較,有非常顯著性差異(P0.01)；而5 J/cm2劑量組與對照組比較,無顯著性差異(P0.05)。Ⅲ型膠原含量,與對照組比較,UVA 20 J/cm2照射組Ⅲ型膠原含量明顯降低,有非常顯著性差異(P0.01),說明成纖維細胞在UVA 20J/cm2照射劑量后,能明顯抑制膠原合成,而UVA 5 J/cm2、UVA 10 J/cm2劑量組與對照組比較,Ⅲ型膠原含量無顯著性差異(P0.05)。2.隨著UVA照射劑量增加,成纖維細胞c.junmRNA表達增加,與對照比較有顯著差異,(p0.01)。c-fosmRNA各組無差異(P0.05)。MMP-1、MMP-3隨著照射劑量增加mRNA表達也增加,有顯著差異。蛋白表達也呈現(xiàn)出與基因表達的一致性。C-jun隨著照射劑量增加而增加,各組間差異p0.01,而c-fos表達無明顯變化(P0.05)。3,設計出5條c-junsiRNA,經(jīng)realtimePCR,及westembolg從基因?qū)用婧偷鞍讓用婀餐瑢ζ浜Y選出沉默效率最高的c-junsiRAN,沉默效率大于80%。序列為：GCAUUCUUGUCACAAUAAATT。4,以15J/cm2輻射劑量照射體外培養(yǎng)的成纖維細胞,2小時后轉(zhuǎn)染c-junsiRNA,轉(zhuǎn)染后24小時,MMP-1、MMP-3 mRNA,與對照組(轉(zhuǎn)染陰性序列組)相比mRNA表達下調(diào),其中MMP-1(P0.01)、MMP-3(P0.05)。Ⅰ、Ⅲ型前膠原mRNA,相比對照組(轉(zhuǎn)染陰性序列組)Ⅰ型前膠原(P0.01),Ⅲ型前膠原(P0.05)。48小時后酶聯(lián)免疫法(ELISA)檢測細胞上清液中MMP-1、MMP-3,Ⅰ、Ⅲ型前膠原含量也呈現(xiàn)出與mRNA表達一致的結果MMP-1(P0.01)、MMP-3(P0.05)、Ⅰ膠原(P0.01)、Ⅲ型膠原(P0.05)。5.UVA照射體外培養(yǎng)的皮膚成纖維細胞EGFRmRNA與蛋白表達呈現(xiàn)一致性,均隨照射劑量增高而增高,5 J/cm2(p0.05),10J/cm2、20J/cm2(P0.01)。UVA照射體外培養(yǎng)的皮膚成纖維細胞導致TGF-β1、IGF-1、KGF分泌下降, UVA 10 J/cm2、UVA 20 J/cm2照射組明顯下降,與對照組比較,有顯著性差異(P0.01)。VEGF分泌升高,UVA 20 J/cm2照射組與對照組比較,有非常顯著性差異(P0.01)。 結論：1.UVA照射體外培養(yǎng)的皮膚成纖維細胞,導致細胞衰老,增殖活性下降；對Ⅰ型、Ⅲ型膠原合成有抑制作用。2.UVA照射體外培養(yǎng)的皮膚成纖維細胞激活MAPK信號傳導通路,其下游基因c-jun上調(diào)明顯,c-fos變化不大,其終末基因MMP-1、MMP-3均上調(diào)。3.經(jīng)realtimePCR,及westernbolg從基因?qū)用婧偷鞍讓用婀餐瑢?條c-junsiRNA篩選,篩選出沉默效率最高的c-junsiRAN,沉默效率大于80%。序列為：GCAUUCUUGUCACAAUAAATT,名稱為JUN-h-825。4.UVA照射體外培養(yǎng)的成纖維細胞2小時后,經(jīng)c-junsiRNA轉(zhuǎn)染后的成纖維細胞,一定程度上MMP-1,MMP-3可以逆轉(zhuǎn)下調(diào),Ⅰ型、Ⅲ型膠原合成減少的趨勢也得到減輕。5.UVA照射后體外培養(yǎng)的皮膚成纖維細胞,EGFR表達明顯增加,細胞上清液中的的細胞因子,TGF-β1、IGF-1.KGF分泌下降；VEGF分泌升高。
[Abstract]:Objective To study the morphological changes of skin fibroblasts cultured in vitro after UVA irradiation and the changes of related cytokines, detect the expression of EGFR, the initiating receptor of MAPK signal transduction, and the expression of c-jun, c-fos, the downstream genes of MAPK signal pathway. Fiber cells were transfected to investigate the effects of down-regulation of c-jun gene downstream of MAPK on MMPs and collagen in skin photoaging.
METHODS: 1. Young adult prepuce fibroblasts were cultured and subcultured. The 5th generation cells were divided into different groups. The control group (0J/cm2) was irradiated with different doses of UVA (5J/cm2, 10J/cm2, 20J/cm2), and the photoaging model of fibroblasts was established. The morphology, RT-PCR and ELISA were observed under light and electron microscopy 24 hours after culture. METHODS: The mRNA expression and protein expression of type I and type III procollagen in cultured skin fibroblasts were measured by ELISA before and after irradiation. The cell viability was measured by MTT assay. 2. UVA (5J/cm210J/cm2, 15J/cm2) was used to irradiate the skin fibroblasts. The control group was set up and the downstream genes of MAPK signal transduction pathway, c-jun, c-fos and terminal gene, MMP-1, were detected by realtime PCR 24 hours after irradiation. MMP-3 mRNA expression. The expression of MMP-1 and MMP-3 in cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA), and the expression of c-jun and c-fos was detected by weasternblog. 3. Five c-junsiRNA, realtime PCR, and westembolg were designed to screen the c-Jun from gene and protein levels. The highest rate of c-junsiRAN was obtained, and the optimal transfection conditions were optimized. 4. The cultured fibroblasts were irradiated with 15J/cm2 radiation, then transfected with c-junsiRNA 2 hours later. MMP-1, MMP-3 mRNA, type I and type III procollagen mRNA were detected by realtime PCR 24 hours after transfection, and MMP-1, MMP-3, type I and type III procollagen mRNA were detected by enzyme-linked immunoassay (ELISA) 48 hours later. Content. 5. Fibroblasts were irradiated with (5J/cm 2, 10J/cm 2, 20J/cm 2) to detect the expression of EGFR, the initiating receptor of MAPK signal transduction pathway, and the changes of TGF-beta 1, IGF-1, KGF and VEGF before and after irradiation.
Results: 1. With the increase of UVA dose, the OD value of MTT of fibroblasts decreased. Compared with the control group, the OD value of UVA 10 J / cm2 and UVA 20 J / cm2 irradiation groups decreased significantly (p0.01). The cumulative absorbance of type I (?) mRNA RT-PCR products decreased with the increase of UVA dose, and UVA 10 J / cm2 and UVA 20 J / cm2 irradiation groups decreased significantly (p0.05, P 0.05). Compared with the control group and the other two groups, the content of collagen type III and the expression of mRNA were significantly lower in the UVA 20 J/cm 2 irradiation group (P 0.01). Compared with the control group (P 0.05, P 0.01), the content of type I collagen in UVA 10J/cm2 and UVA 20J/cm2 irradiation groups decreased significantly, indicating that fibroblasts irradiated with UVA 10J/cm2 and UVA 20J/cm2 could inhibit collagen synthesis, and the effect of UVA 20J/cm2 irradiation on collagen synthesis was more obvious than that of control group (P 0.05, P 0.01). There was a significant difference between the control group and the other two groups (P 0.01), but there was no significant difference between the 5 J / cm 2 dose group and the control group (P 0.05). The content of collagen type III in the UVA 20 J / cm 2 irradiation group was significantly lower than that in the control group (P 0.01), indicating that fibroblasts were significantly lower than that in the UVA 20 J / cm 2 irradiation group (P 0.01). Compared with the control group, the content of collagen type III was not significantly different (P 0.05). 2. With the increase of UVA dose, the expression of C. Jun mRNA in fibroblasts increased significantly (P 0.01). There was no significant difference in the expression of C - fos mRNA between the groups (P 0.05). MMP - 1 and MMP - 3 with the increase of UVA dose. The expression of C-jun increased with the increase of irradiation dose, but the expression of c-fos did not change significantly (P 0.05). 3. Five c-junsiRNA were designed and screened for silencing by realtime PCR and westembolg at both gene and protein levels. The most efficient c-junsiRAN, the silencing efficiency was more than 80%. The sequence was GCAUUCUUGUCACA AAAATT.4, irradiated with 15J/cm 2 radiation dose, transfected with c-junsiRNA 2 hours later, 24 hours after transfection, MMP-1, MMP-3 mRNA expression was down-regulated compared with the control group (transfection negative sequence group), MMP-1 (P 0.01), MMP-3 (P 0.05). MMP-1, MMP-3, type I and type III procollagen in supernatant were detected by enzyme-linked immunosorbent assay (ELISA) 48 hours after transfection. MMP-1 (P 0.01), MMP-3 (P 0.05), type I collagen (P 0.01), type III collagen (P 0.05) and type I procollagen (P 0.05) were also detected by ELISA. 5. The expression of EGFR mRNA and protein in cultured skin fibroblasts irradiated by UVA was consistent with that in vitro. Both EGFR mRNA and protein increased with the increase of irradiation dose. 5 J/cm2 (p0.05), 10 J/cm2, 20 J/cm2 (P 0.01). Cultured skin fibroblasts irradiated by UVA decreased the secretion of TGF-beta 1, IGF-1, KGF, UVA 10 J/cm2 and UVA 20 J/cm2, and decreased significantly compared with the control group. Compared with the control group, there was a significant difference (P 0.01). The secretion of VEGF was elevated. There was a significant difference (P 0.01) between the UVA 20 J/cm 2 irradiation group and the control group.
Conclusion: 1. UVA irradiation of cultured skin fibroblasts can induce cell senescence and decrease proliferation activity, and inhibit the synthesis of collagen type I and type III. 2. UVA irradiation of cultured skin fibroblasts can activate MAPK signal transduction pathway. The downstream genes c-Jun are up-regulated and c-fos is not changed, and the terminal genes MMP-1 and MMP-3 are both up-regulated. Five c-junsiRANs were screened by realtime PCR and Western bolg at the gene and protein levels. The highest silencing efficiency of c-junsiRAN was screened out. The sequence was GCAU UCU UCA CAA UAAATT, named JUN-h-825.4. After 2 hours of UVA irradiation, fibroblasts were transfected with c-junsiRNA. Vitamin cells, to a certain extent, MMP-1 and MMP-3 can reverse the downregulation, and the decrease of collagen synthesis of type I and type III can also be alleviated. 5. After UVA irradiation, the expression of EGFR in cultured skin fibroblasts increased significantly, the secretion of cytokines, TGF-beta 1 and IGF-1.KGF in cell supernatant decreased, and the secretion of VEGF increased.
【學位授予單位】：昆明醫(yī)學院
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R758.1
本文編號：2245165
[Abstract]:Objective To study the morphological changes of skin fibroblasts cultured in vitro after UVA irradiation and the changes of related cytokines, detect the expression of EGFR, the initiating receptor of MAPK signal transduction, and the expression of c-jun, c-fos, the downstream genes of MAPK signal pathway. Fiber cells were transfected to investigate the effects of down-regulation of c-jun gene downstream of MAPK on MMPs and collagen in skin photoaging.
METHODS: 1. Young adult prepuce fibroblasts were cultured and subcultured. The 5th generation cells were divided into different groups. The control group (0J/cm2) was irradiated with different doses of UVA (5J/cm2, 10J/cm2, 20J/cm2), and the photoaging model of fibroblasts was established. The morphology, RT-PCR and ELISA were observed under light and electron microscopy 24 hours after culture. METHODS: The mRNA expression and protein expression of type I and type III procollagen in cultured skin fibroblasts were measured by ELISA before and after irradiation. The cell viability was measured by MTT assay. 2. UVA (5J/cm210J/cm2, 15J/cm2) was used to irradiate the skin fibroblasts. The control group was set up and the downstream genes of MAPK signal transduction pathway, c-jun, c-fos and terminal gene, MMP-1, were detected by realtime PCR 24 hours after irradiation. MMP-3 mRNA expression. The expression of MMP-1 and MMP-3 in cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA), and the expression of c-jun and c-fos was detected by weasternblog. 3. Five c-junsiRNA, realtime PCR, and westembolg were designed to screen the c-Jun from gene and protein levels. The highest rate of c-junsiRAN was obtained, and the optimal transfection conditions were optimized. 4. The cultured fibroblasts were irradiated with 15J/cm2 radiation, then transfected with c-junsiRNA 2 hours later. MMP-1, MMP-3 mRNA, type I and type III procollagen mRNA were detected by realtime PCR 24 hours after transfection, and MMP-1, MMP-3, type I and type III procollagen mRNA were detected by enzyme-linked immunoassay (ELISA) 48 hours later. Content. 5. Fibroblasts were irradiated with (5J/cm 2, 10J/cm 2, 20J/cm 2) to detect the expression of EGFR, the initiating receptor of MAPK signal transduction pathway, and the changes of TGF-beta 1, IGF-1, KGF and VEGF before and after irradiation.
Results: 1. With the increase of UVA dose, the OD value of MTT of fibroblasts decreased. Compared with the control group, the OD value of UVA 10 J / cm2 and UVA 20 J / cm2 irradiation groups decreased significantly (p0.01). The cumulative absorbance of type I (?) mRNA RT-PCR products decreased with the increase of UVA dose, and UVA 10 J / cm2 and UVA 20 J / cm2 irradiation groups decreased significantly (p0.05, P 0.05). Compared with the control group and the other two groups, the content of collagen type III and the expression of mRNA were significantly lower in the UVA 20 J/cm 2 irradiation group (P 0.01). Compared with the control group (P 0.05, P 0.01), the content of type I collagen in UVA 10J/cm2 and UVA 20J/cm2 irradiation groups decreased significantly, indicating that fibroblasts irradiated with UVA 10J/cm2 and UVA 20J/cm2 could inhibit collagen synthesis, and the effect of UVA 20J/cm2 irradiation on collagen synthesis was more obvious than that of control group (P 0.05, P 0.01). There was a significant difference between the control group and the other two groups (P 0.01), but there was no significant difference between the 5 J / cm 2 dose group and the control group (P 0.05). The content of collagen type III in the UVA 20 J / cm 2 irradiation group was significantly lower than that in the control group (P 0.01), indicating that fibroblasts were significantly lower than that in the UVA 20 J / cm 2 irradiation group (P 0.01). Compared with the control group, the content of collagen type III was not significantly different (P 0.05). 2. With the increase of UVA dose, the expression of C. Jun mRNA in fibroblasts increased significantly (P 0.01). There was no significant difference in the expression of C - fos mRNA between the groups (P 0.05). MMP - 1 and MMP - 3 with the increase of UVA dose. The expression of C-jun increased with the increase of irradiation dose, but the expression of c-fos did not change significantly (P 0.05). 3. Five c-junsiRNA were designed and screened for silencing by realtime PCR and westembolg at both gene and protein levels. The most efficient c-junsiRAN, the silencing efficiency was more than 80%. The sequence was GCAUUCUUGUCACA AAAATT.4, irradiated with 15J/cm 2 radiation dose, transfected with c-junsiRNA 2 hours later, 24 hours after transfection, MMP-1, MMP-3 mRNA expression was down-regulated compared with the control group (transfection negative sequence group), MMP-1 (P 0.01), MMP-3 (P 0.05). MMP-1, MMP-3, type I and type III procollagen in supernatant were detected by enzyme-linked immunosorbent assay (ELISA) 48 hours after transfection. MMP-1 (P 0.01), MMP-3 (P 0.05), type I collagen (P 0.01), type III collagen (P 0.05) and type I procollagen (P 0.05) were also detected by ELISA. 5. The expression of EGFR mRNA and protein in cultured skin fibroblasts irradiated by UVA was consistent with that in vitro. Both EGFR mRNA and protein increased with the increase of irradiation dose. 5 J/cm2 (p0.05), 10 J/cm2, 20 J/cm2 (P 0.01). Cultured skin fibroblasts irradiated by UVA decreased the secretion of TGF-beta 1, IGF-1, KGF, UVA 10 J/cm2 and UVA 20 J/cm2, and decreased significantly compared with the control group. Compared with the control group, there was a significant difference (P 0.01). The secretion of VEGF was elevated. There was a significant difference (P 0.01) between the UVA 20 J/cm 2 irradiation group and the control group.
Conclusion: 1. UVA irradiation of cultured skin fibroblasts can induce cell senescence and decrease proliferation activity, and inhibit the synthesis of collagen type I and type III. 2. UVA irradiation of cultured skin fibroblasts can activate MAPK signal transduction pathway. The downstream genes c-Jun are up-regulated and c-fos is not changed, and the terminal genes MMP-1 and MMP-3 are both up-regulated. Five c-junsiRANs were screened by realtime PCR and Western bolg at the gene and protein levels. The highest silencing efficiency of c-junsiRAN was screened out. The sequence was GCAU UCU UCA CAA UAAATT, named JUN-h-825.4. After 2 hours of UVA irradiation, fibroblasts were transfected with c-junsiRNA. Vitamin cells, to a certain extent, MMP-1 and MMP-3 can reverse the downregulation, and the decrease of collagen synthesis of type I and type III can also be alleviated. 5. After UVA irradiation, the expression of EGFR in cultured skin fibroblasts increased significantly, the secretion of cytokines, TGF-beta 1 and IGF-1.KGF in cell supernatant decreased, and the secretion of VEGF increased.
【學位授予單位】：昆明醫(yī)學院
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R758.1
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